Heteroaryl substituted benzothiazole dioxetanes

ABSTRACT

Chemiluminescent heteroaryl substituted benzothiazole 1,2-dioxetane compounds capable of producing light energy when decomposed are provided. These chemiluminescent compounds are represented by the general formula: 
                 
 
The heteroaryl substituent Y can be, for example, a pyridyl group or a benzothiazolyl group. The heteroaryl substituted benzothiazole compounds are substantially stable at room temperature. Kits including the heteroaryl substituted dioxetane compounds as well as methods for using these compounds for detecting the presence of one or more analytes in a sample are also provided.

This Application claims the benefit of Provisional Application No.60/094,336, filed Jul. 28, 1998.

This application is a Continuation of application Ser. No. 09/945,652filed on Sep. 5, 2001, now U.S. Pat. No. 6,660,529, which isContinuation-In-Part of U.S. Ser. No. 09/362,047 filed Jul. 28, 1999,now U.S. Pat. No. 6,359,441.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to improved chemiluminescent 1,2-dioxetanecompounds. More particularly, this invention relates to improvedenzymatically cleavable chemiluminescent 1,2-dioxetane compounds thatcontain enzymatically removable labile groups. Such labile groupsprevent the molecule from decomposing to produce light, i.e., visiblelight or light detectable by appropriate instrumentation, until anappropriate enzyme is added to remove the labile group.

One enzyme molecule can affect the removal, through a catalytic cycle,of its complimentary labile group from thousands of enzymaticallycleavable chemiluminescent 1,2-dioxetane molecules. This is a markedcontrast to the situation with chemically cleavable chemiluminescent1,2-dioxetanes, where one molecule of a chemical cleaving agent isneeded to remove the complimentary labile group from each dioxetanemolecule.

Enzymnatically cleavable light-producing 1,2-dioxetane compounds willusually also contain stabilizing groups, such as an adamantylidene groupspiro bonded to the dioxetane ring's 3-carbon atom, that will aid inpreventing the dioxetane compound from undergoing spontaneousdecomposition at room temperature (about 25° C.) before the bond bywhich the enzymatically cleavable labile group is attached to theremainder of the molecule is intentionally cleaved. Wieringa, et al.,Tetahedron Letters, 169 (1972), and McCapra, et al., J. Chem. Soc.,Chem. Comm., 944 (1977). These stabilizing groups thus permit suchdioxetanes to be stored for exceptionally long periods of time beforeuse, e.g., for from about 12 months to as much as about 12 years attemperatures ranging from about 40° C. to about as much as 30° C.,without undergoing substantial decomposition.

This invention further relates to the incorporation of its dioxetanemolecules in art-recognized immunoassays, chemical assays and nucleicacid probe assays, and to their use as direct chemical/physical probesfor studying the molecular structure or microstructures of variousmacromolecules, synthetic polymers, proteins, nucleic acids, catalyticantibodies, and the like, to permit an analyte (e.g. a chemical orbiological substance whose presence, amount or structure is beingdetermined) to be identified or quantified.

2. Background of the Invention

The use of 1,2-dioxetanes as chemiluminescent compounds is wellestablished. These compounds, for example, have been used as reportersand labels in ultra sensitive assays for the detection of a variety ofbiological materials. By using 1,2-dioxetanes, these assays can beconducted quickly and without resort to exotic conditions or elaborateapparatus. See, for example, U.S. Pat. Nos. 4,931,223; 4,931,569;4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772; 5,220,005;5,225,584; 5,326,882; 5,330,900; 5,336,596; and 5,871,938. All of theforegoing are incorporated herein by reference. Other patents commonlyassigned with this application have issued, and other applications arepending. Together, this wealth of patent literature addresses1,2-dioxetanes stabilized by a typically polycyclic group, such as anadamantyl group spiro-bonded to one of the carbons of the dioxetane ringand another moiety (e.g., an aryl group) bonded to the remainder carbonof the dioxetane ring. This moiety is typically electron sensitive.Deprotection of the electron sensitive moiety results in the formationof an anion, generally an oxyanion, which is unstable and decomposes.Through decomposition, the O—O bond in the dioxetane is broken and aphoton is generated. The same carbon atom to which this electronsensitive moiety is bonded may bear an alkoxy or other electron-activegroup.

The first of the dioxetanes of this class to be commercialized was3-(4-methoxy-spiro(1,2-dioxetane-3,2′-tricyclo(3.3.1.1^(3.7))decan)-4-yl)phenyl phosphate, particularly the disodium salt, generallyknown as AMPPD®. This compound has been commercialized by the assigneeof this application, Tropix, Inc. of Bedford, Mass., as well as Lumigen,Inc. of Detroit, Mich. Superior performance of the above-describedcompounds can be obtained by selective substitution on the spiro-boundadamantane ring. For example, substitution at either bridgehead carbonwith an electron active species, such as chlorine, has been found toimprove reaction speed and signal to noise ratio (S/N). The chlorinesubstituted counterpart of AMPPD®, available under the trademark CSPD®,has also been widely commercialized by Tropix. “Third-generation”dioxetane compounds of similar structure, wherein the aryl moiety alsobears an electron active substituent, such as chlorine, have been foundto afford further improvements in performance. The 1,2-dioxetanes havingaryl groups bearing phosphate moieties are available under thetrademarks CDP® and CDP-Star®, both of which are registered trademarksof Tropix, Inc.

Various materials have been used to enhance the chemiluminescentemissions of 1,2-dioxetanes. These materials, commonly referred to aschemiluminescent enhancing agents, include polymeric ammonium,phosphonium or sulphonium salts such as poly[vinyl benzyl(benzyldimethylammonium chloride)] (“BDMQ”) and other hetero polar polymers.

It has been observed, however, that chemiluminescent dioxetanes such asAMPPD® in aqueous solution and also in the present chemiluminescentenhancers, may exhibit longer than optimum periods of time to reachconstant light emission characteristics. The half-life or “t_(1/2) ” ofthe active chemiluminescent species is defined as the time necessary toobtain one-half of the maximum chemiluminescence intensity at constant,steady-state light emission levels. This emission half-life can vary asa function of the stability of the dioxetane oxyanion in variousenvironments. For example, the half-life of AMPPD® at concentrationsabove 2×10⁻⁵ M in an aqueous solution at a pH 9.5 in the presence ofBDMQ has been found to be approximately 7.5 minutes. At 4×10⁻³ M in theabsence of BDMQ, the t_(1/2) of AMPPD® has been found to beapproximately 30-60 minutes, while at 2×10⁻⁵ M in an aqueous solution,the t_(1/2) of AMPPD® has been found to be about 2.5 minutes.

Chemiluminescent intensity is typically measured after achieving steadystate light emission kinetics. Statistically, approximately sevent_(1/2) periods are required to reach steady-light emission kinetics.While chemiluminescent intensity can be measured before achieving steadystate kinetics, sophisticated thermally-controlled luminometryinstrumentation must be used if one wishes to acquire precise data priorto achieving steady-state emission kinetics. Therefore, in assays suchas bioassays that employ enzymatically cleavable chemiluminescent1,2-dioxetanes as reporter molecules, it is desirable to reachsteady-state light emission kinetics as quickly as possible.

Furthermore, AMPPD®, in an aqueous buffered solution both in thepresence and absence of chemiluminescent enhancers such as BDMQ,exhibits higher than desirable thermal and non-enzymatically activatedlight emission, or “noise.” Such noise can be attributed to emissionfrom the excited state adamantanone and of the methyl m-oxybenzoateanion derived from the aromatic portion of the AMPPD® molecule. Themeasured noise level of AMPPD® can be as much as two orders of magnitudeabove the dark current in a standard luminometer. This noise cantherefore limit the levels of detection and prevent the realization ofultimate sensitivity in chemiluminescent assays.

Also, various instruments for detecting chemiluminescent emission suchas charge coupled device (CCD) cameras have greater detectionsensitivities in the green and red wavelengths. AMPPD® and relateddioxetanes typically emit in the shorter wavelengths (e.g., bluewavelengths) of the visible spectrum. Heretofore, polymeric enhancershave been used to “shift” the emission wavelength toward the green orred end of the visible spectrum. It would therefore be desirable toobtain dioxetanes which emit radiation in wavelengths closer to thegreen portion or toward the red end of the visible spectrum to enhancedetection sensitivity.

It is therefore an object of this invention to decrease the timenecessary to conduct assays, and particularly bioassays, in whichenzymatically cleavable chemiluminescent 1,2-dioxetanes are used asreporter molecules.

It is also an object of this invention to provide new and improvedenzymatically cleavable chemiluminescent 1,2-dioxetanes which, when usedas reporter molecules in assays, and in particular bioassays, reduce thetime required to complete the assay.

A further object of this invention is to provide new and improvedenzymatically cleavable chemiluminescent 1,2-dioxetanes for use assubstrates for enzyme-based assays, and particularly bioassays, whichprovide improved signal to background behavior and thus provide improveddetection levels.

A further object of this invention is the provision of dioxetane whoseemission wavelengths are shifted toward the green and red wavelengths.

A still further object of this invention is to provide novelintermediates useful in synthesizing these improved enzymaticallycleavable 1,2-dioxetanes.

Another object of this invention is to provide methods of preparingthese enzymatically cleavable chemiluminescent 1,2-dioxetanes andintermediates thereof.

These and other objects, as well as the nature, scope and utilization ofthis invention, will become readily apparent to those skilled in the artfrom the following description and the appended claims.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, a chemiluminescentcompound of the general formula set forth below is provided.

Each of the substituents R in the above formula may independently be abranched alkyl or cycloalkyl group which provides stabilization for thedioxetane. Alternatively, both R groups may be joined in a cycloalkyl orpolycycloalkyl moiety spiro bound to the dioxetane ring. Each of the Rgroups or the spiro bound cyclic group may be unsubstituted orsubstituted with a halogen atom, an alkoxy group, or anelectron-withdrawing organic group. The substituent R¹ can be an arylgroup or an alkyl group of 1-20 carbon atoms, which may be optionallysubstituted with one or more halogen atoms. The substituent X may be anyprotecting group which is removable by chemical or enzymatic means.According to a preferred embodiment of the invention, Y is aheterocyclic aromatic moiety (i.e., a heteroaryl group). According to afurther preferred embodiment of the invention, the heterocyclic aromaticmoiety Y is: 2-, 3-, or 4-pyridyl; 2-benzothiazolyl; 2-benzoxazolyl;2-benzofuranyl; 2-benzothienyl; 2-, 4-, or 5-thiazolyl; 3-, 4-, 5-, 6-,or 7-quinolinyl; 3-, 4-, or 5-isoxazolyl; 3-, 4-, or 5 -isothiazolyl; or2-, 4-, 5-, or 6-pyrimidinyl.

According to a second aspect of the invention, a kit for detecting thepresence of an analyte in a sample is provided. The kit includes the1,2-dioxetane compound set forth above. The kit according to theinvention can further include a substance such as an enzyme which, inthe presence of the dioxetane compound, causes the dioxetane compound todecompose and generate light.

According to a third aspect of the invention, a method for detecting thepresence of an analyte in a sample is provided. The method includesadding the dioxetane compound set forth above to the sample wherein themoiety X can be removed by the analyte. The method further includesincubating the sample and inspecting the sample for generated light. Thepresence of generated light indicates the presence of the substance. Theamount of light detected can be used to determine the amount of thesubstance present in the sample. Methods for simultaneously detectingthe presence of two or more analytes in a sample are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of a benzothiazole 1,2-dioxetanephosphate (BZPD);

FIG. 2A shows a first set of steps for the synthesis of the fusedbenzothiazole 1,2-dioxetane phosphate (BZPD) of FIG. 1 including thesynthesis of a benzothiazole compound with a phenyl substituent;

FIG. 2B shows a second set of steps for the synthesis of the fusedbenzothiazole 1,2-dioxetane phosphate (BZPD) of FIG. 1 including thesynthesis of the corresponding benzothiazole enol ether phosphate;

FIG. 3 shows a final step for the synthesis of the fused benzothiazole1,2-dioxetane phosphate (BZPD) of FIG. 1 including a step ofphotoxygenation of the corresponding benzothiazole enol ether phosphate;

FIG. 4 illustrates the synthesis of a benzothiazole compound with anaryl substituent;

FIG. 5 illustrates the synthesis of a benzothiazole enol etherderivative with a benzyloxy linker for attachment of a biological moietyor an energy transfer fluorophore;

FIG. 6 shows a benzothiazole-DROX energy transfer enzyme substrate;

FIG. 7 shows the emission spectrum of the dioxetane of FIG. 1 in thepresence of alkaline phosphatase at a pH of 10;

FIG. 8 shows the emission spectrum of the dioxetane of FIG. 1 in thepresence of alkaline phosphatase at a pH of 10;

FIG. 9A shows luminescence (RLU) values for CSPD®, CDP-Star® and BZPD ata pH of 8.5 and at a time of 4 min.;

FIG. 9B shows the signal to noise ratio (S/N) for the luminescence ofCSPD®, CDP-Star® and BZPD at a pH of 8.5 and at a time of 4 min.;

FIG. 10A shows luminescence (RLU) values for CSPD®, CDP-Star® and BZPDat a pH of 8.5 and at a time of 24 min.;

FIG. 10B shows the signal to noise ratio (S/N) for the luminescence ofCSPD®, CDP-Star® and BZPD at a pH of 8.5 and at a time of 24 min.;

FIG. 11A is a graph showing luminescence (RLU) as a function of alkalinephosphatase concentration for BZPD compared to CSPD® and CDP-Star® at apH of 8.5 and at a time of 4 min.;

FIG. 11B is a graph showing signal to noise ratio (S/N) as a function ofalkaline phosphatase concentration for the luminescence of BZPD comparedto CSPD® and CDP-Star® at a pH of 8.5 and at a time of 4 min.;

FIG. 12A is a graph showing luminescence (RLU) as a function of alkalinephosphatase concentration for BZPD compared to CSPD® and CDP-Star® at apH of 8.5 and at a time of 24 min.;

FIG. 12B is a graph showing signal to noise ratio (S/N) as a function ofalkaline phosphatase concentration for the luminescence of BZPD comparedto CSPD® and CDP-Star® at a pH of 8.5 and at a time of 24 min.;

FIG. 13 shows the chemical structure of a 3-pyridyl substitutedbenzothiazole 1,2-dioxetane according to the invention;

FIG. 14 shows the general structure of a 2-benzothiazolyl substitutedbenzothiazole dioxetane according to a preferred embodiment of theinvention;

FIG. 15 shows the general structure of a second embodiment of aheteroaryl substituted benzothiazole dioxetane compound according to theinvention;

FIG. 16 shows a first route for the synthesis of a 3-pyridyl substitutedbenzothiazole 1,2-dioxetane; and

FIG. 17 shows a second route for the synthesis of a 3-pyridylsubstituted benzothiazole 1,2-dioxetane.

DETAILED DESCRIPTION

We now describe the structure, synthesis, and use of preferredembodiments of the present invention.

According to the invention, a chemiluminescent compound having thegeneral formula (I) is provided:

In general formula (I) above, each R may independently be any branchedalkyl or cycloalkyl group which provides stabilization for thedioxetane. Alternatively, both R groups together can form a cycloalkylor polycycloalkyl moiety spiro bound to the dioxetane ring. Each of theR groups may be substituted or unsubstituted. Further, if the R groupsform a spiro bound cyclic moiety, the spiro bound cyclic moiety may besubstituted or unsubstituted. The R groups or the spiro bound cyclicgroup may be substituted, for example, with a halogen atom, an alkoxygroup, or an electron-withdrawing organic group. The substituent R¹ canbe an aryl group or an alkyl group having 1-20 carbon atoms, which maybe optionally substituted with one or more halogen atoms. According tothe invention, the substituent Y on the 2-carbon of the benzothiazolemoiety is a heteroaryl group. The heteroaryl group can be substitutedwith electron active groups or solubilizing groups. The substituent Xmay be any protecting group which is removed by chemical or enzymaticmeans.

According to a second aspect of the invention, a chemiluminescentcompound having the general formula (II) is provided:

The substituents X and Y in Formula II are defined as set forth abovefor Formula I. The substituents R²-R⁶ in Formula (II) above canindependently be hydrogen, alkyl, substituted alkyl, aryl, orsubstituted aryl. Further, the substituents R³ and R⁴ may be joined as aspiro-fused cycloalkyl group.

The dioxetanes according to Formula II of the invention can besynthesized by way of the corresponding benzothiazole aldehyde ortoluene derivative using permanganate oxidation to provide a carboxylicacid. Esters of this carboxylic acid can then be used to preparedioxetane precursors for the above compounds. Methods of this type aredescribed in U.S. Pat. No. 5,731,445, which is incorporated herein byreference.

Representative identities for each of the substituents of formulas I andII will be familiar to those of skill in the art, given the novelchemical formulas herein and the earlier patents of Tropix incorporatedherein by reference. Preferred identities for R include straight orbranched chain alkyls of 2-12 carbon atoms. Branched alkyl groups arepreferred. Further, each alkyl group may be substituted with one or moreelectron-withdrawing or electron-donating groups, and/or each alkylmoiety R may be substituted with one or more groups which increase thesolubility of the overall dioxetane, which is generally quitehydrophobic. Preferred solubilizing groups include carboxylic acidmoieties, sulfonic acid moieties, phosphoric acid moieties, ammoniummoieties, etc.

In a preferred embodiment, both R groups together form a spiro boundadamantyl group, which may be unsubstituted or substituted at either orboth bridgehead carbons with an electron-active (i.e., anelectron-withdrawing or an electron-donating) group. Suitable electronactive groups include alkoxy groups having 1-7 carbon atoms, halogroups, alkyl groups, etc. Exemplary substituents on the adamantyl groupare set forth in U.S. Pat. No. 5,112,960, which is incorporated hereinby reference. Further, the identity of each group R can be selected soas to provide steric stabilization for the dioxetane to preventpremature decomposition.

The substituents R²-R⁶ can each be independently selected with theexception that R³ and R⁴ may be joined to form a spiro-fused cycloalkylgroup, as described above for the R groups of Formula I. Otherwise,R²-R⁶ can be independently selected from hydrogen, alkyl groups of 1-6carbon atoms which are unsubstituted or substituted with one or morehalogen groups (e.g., trifluroalkyl), hydroxy, phenyl, naphthyl, etc.Any of the moieties R²-R⁶, either alone or in combination, may befurther substituted with one or more groups calculated to enhance thewater solubility of the dioxetane, as described above. Further, eachmoiety R²-R⁶ may bear one or two water solubility-enhancing groups.Instead of alkyl, each and/or all of R²-R⁶ may be an aryl group,preferably phenyl.

The heteroaryl group Y can be substituted with electron active groups.Preferred electron-active substituents on Y include chloro, aralkenyl,alkoxy (—OR), aryloxy (—OAr), trialkylammonium (—NR₃+), alkylamido(—NHCOR, —NRCOR′), arylamido (—NHCOAr, —NRCOAr, —NarCOAr), arylcarbamoyl(—NHCOOAr, —NRCOOAr), alkylcarbamoyl (—NHCOOR, —NRCOOR′), cyano (—CN),nitro (—NO₂), ester (—COOR, —COOAr), alkyl- or arylsulfonamido (—NHSO₂R,—NHSO₂Ar), trifluoromethyl (—CF₃), aryl (—Ar), alkyl (—R), trialkyl-,triaryl-, or alkylarysilyl (—SiR₃, —SiAr₃, —SiArR₂), alkyl- orarylamidosulfonyl (—SO₂NHCOR, —SO₂NHCOAr), alkyl- or arylsulfonyl(—SO₂Ar), alkyl- or arylthioethers (—SR, —SAr). The size of the Zsubstituent is generally limited only by solubility concerns. Wherereference is made to alkyl or R, R′, etc., the alkyl moiety preferablyhas from 1-12 carbon atoms. Suitable aryl moieties include phenyl andnaphthyl. Particularly preferred electron active substituents includechloro and alkoxy.

The heteroaryl group Y can also be substituted with water solubilizinggroups such as carboxylic acids, sulfates, sulfonates, phosphates, andammonium groups. The water solubility of Y can also be increased byalkylation of one of the heteroatoms in Y to produce a quaternizedheteroaryl group.

As set forth in the aforementioned U.S. Pat. No. 5,538,847,electron-donating groups, such as methoxy groups, enhance the aniondecomposition process, whereas electron-withdrawing groups, such aschlorine, may retard the same decomposition reaction. Surprisingly, theinfluence of substituents on the aryl ring may have the opposite effectto substituents on the adamantyl group or other steric stabilizinggroup.

According to the invention, Y is an aromatic heterocyclic (e.g.,heteroaryl) group. Particularly suitable heteroaryl groups include 2-,3-, or 4-pyridyl; 2-benzothiazolyl; 2-benzoxazolyl; 2-benzofuranyl;2-benzothienyl; 2-, 4-, or 5-thiazolyl; 3-, 4-, or 5-isoxazolyl; 3-, 4-,or 5-isothiazolyl; 3-, 4-, 5-, 6- or 7 -quinolinyl; or 2-, 4-, 5-, or6-pyrimidinyl. The heteroaryl group Y according to the invention can beeither unsubstituted or substituted. Preferred substituents for theheteroaryl group include one or more electron active groups.

Typically, the X substituent in Formulae I and II above is anenzyme-labile group. Although preferred enzyme-labile groups includephosphate moieties and galactoside moieties, virtually anyenzyme-cleavable group, which, upon cleavage, leaves an oxyanion, issuitable for use in the invention. A large variety of enzyme-cleavablegroups are set forth in U.S. Pat. No. 5,605,795, which is incorporatedherein by reference. In general, in addition to the phosphate esters,moiety X may be any of the moieties identified for group Z in U.S. Pat.No. 5,605,795, incorporated herein by reference, including substratesfor esterases, decarboxylases, phospholipases, α- or β-xylosidase,fucosidases, glucosidases, and thioglucosidases, galactosidases,mannosidases, fructofuranosidases, glucosiduronases, trypsin, etc.Additionally, the moiety —OX can be replaced by any of a wide variety ofpeptides cleavable by proteolytic enzymes, such as those set forth inU.S. Pat. No. 5,591,591, which is incorporated herein by reference.

As previously noted, there are situations where non-enzymatic chemicaltriggering, as opposed to enzymatic triggering, may be desired. In theseinstances, the X substituent can be, for example, H, trialkylsilyl, etc.Chemical triggering, as well as various identities for the substituentX, are disclosed in U.S. Pat. No. 5,652,345, which is also incorporatedherein by reference.

In monitoring, and measuring (e.g., quantifying) chemiluminescence, awide variety of apparatuses have been developed. Among the mostsensitive, and particularly suited to high throughput screeningapplications and the like, are CCD cameras. Typical luminescent emissionfrom dioxetanes is in the blue wavelengths of the visible spectrum. CCDcameras, however, can have difficulty registering blue emissions.Therefore, only the “edge” of the longer wavelengths of the blueemission are typically observed by the camera. By employing abenzothiazole resonating moiety on the dioxetane according to theinvention, the light being emitted can be green-shifted. That is, theemission can be shifted toward the green or red region of the visiblespectrum.

Prior art dioxetanes are typically used with enhancement agents whichact to sequester the dioxetane in hydrophobic regions thus reducing thechemiluminescent quenching that can be observed in the presence ofwater. These enhancement molecules are preferably onium quaternarypolymers such as phosphonium, sulfonium and ammonium polymers.Representative polymers, and their effects, are set forth in U.S. Pat.No. 5,330,900, which is incorporated herein by reference. These polymersmay be used alone, or together with a surfactant additive, to furtherimprove the enhancement value, as disclosed in U.S. Pat. No. 5,547,836,which is also incorporated herein by reference.

In the present invention, as a result of the green shifting of thedioxetane emission, and the enhanced hydrophobicity of the dioxetanesdue to the presence of the benzothiazole, lower concentrations ofenhancement agents and other additives can be used.

Referring more particularly to the figures, FIG. 1 shows the generalformula for the chemical structure of benzothiazole dioxetane phosphate.This molecule is referred to herein as BZPD. The BZPD molecule shown inFIG. 1 has a phenyl substituent at the 2 position of the benzothiazolegroup. According to the invention, however, other substituents can beused on the benzothiazole. Preferred substituents according to theinvention are aromatic heterocyclic (e.g. heteroaryl) groups such aspyridyl, benzothiazolyl, benzoxazolyl, etc. According to a preferredembodiment of the invention, the heterocyclic aromatic moiety is 2-, 3-,or 4-pyridyl; 2-benzothiazolyl; 2-benzoxazolyl; 2-benzofuranyl;2-benzothienyl; 2-, 4-, or 5-thiazolyl; 3-, 4-, or 5-isoxazolyl; 3-, 4-,or 5-isothiazolyl; 3-, 4-, 5-, 6-, or 7-quinolinyl; or 2-, 4-, 5-, or6-pyrimidinyl.

The synthesis methods illustrated in FIGS. 2-6 and described below aredirected to a BZPD dioxetane with a phenyl or substituted phenylsubstituent. However, these techniques could be adapted to synthesize asimilar dioxetane molecule having a heteroaryl substituent on thebenzothiazole group according to the invention. Adaptation of thetechniques illustrated in FIGS. 2-6 would be within the level ofordinary skill in the art.

The synthesis of the BZPD dioxetane of FIG. 1 is illustrated in FIGS.2A, 2B and 3. FIG. 2A shows a first set of steps for the synthesis ofthe fused benzothiazole 1,2-dioxetane phosphate (BZPD) of FIG. 1including the synthesis of a benzothiazole compound with a phenylsubstituent. FIG. 2B shows a second set of steps for the synthesis ofthe fused benzothiazole 1,2-dioxetane phosphate (BZPD) of FIG. 1including the synthesis of the corresponding benzothiazole enol etherphosphate. FIG. 3 shows a final step for the synthesis of the fusedbenzothiazole 1,2-dioxetane phosphate (BZPD) of FIG. 1 including a stepof photooxygenation of the corresponding benzothiazole enol etherphosphate.

Although conventional starting materials may be used to synthesize thedioxetanes of the present invention, novel isothiocyanates are set forthas an aspect of the invention and are illustrated in FIG. 4 wherein thesynthesis of a benzothiazole compound with an aryl substituent isillustrated.

FIG. 5 illustrates the synthesis of a benzyloxy benzothiazole enol etherderivative which can be used to synthesize a dioxetane having a site forattachment of a biological moiety or an energy transfer fluorophoreaccording to the invention.

FIG. 6 shows a benzothiazole-DROX energy transfer enzyme substrate.

FIGS. 7 and 8 show the intensity of emissions as a function ofwavelength for BZPD. FIG. 7 illustrates the “green shifting” of thewavelength emission of BZPD, showing a peak wavelength above 550 nm, inthe absence of any enhancement agent. FIG. 8 illustrates that, in thepresence of an enhancement agent (i.e., Sapphire-II™), long wavelengthintensity of the chemiluminescent emissions can be further enhanced.

The relative chemiluminescent performance of BZPD is compared with othercommercially available dioxetanes in FIGS. 9-12. FIG. 9A showsluminscence (RLU) at a pH of 8.5 and at a time of 4 minutes for BZPDcompared to CSPD® (a phenyl dioxetane bearing a chlorine substituent onthe adamantyl group) and CDP-Star® (a phenyl dioxetane bearing chlorinesubstituents on both the adamantyl group and on the phenyl moiety). Ascan be seen from FIG. 9A, the half-life (t_(1/2)) of BZPD is shorterthan the corresponding half-life of CSPD® or CDP-Star®. As shown in FIG.9B, the inventive dioxetanes also have an excellent signal to noiseratio (S/N) under these conditions. The signal to noise ratio (S/N)values are important because if the background noise is too high (i.e.,if the S/N is too low) the assay can be relatively insensitive no matterhow rapidly the peak intensity is developed.

FIGS. 10A and 10B show the luminescence value and S/N after at a pH of8.5 and at a time of 24 minutes. As can be seen from these figures, BZPDexhibits a significantly higher luminescence and a significantly highersensitivity (S/N) under these conditions.

Sensitivity can be an important characteristic of dioxetane detectionagents. In FIGS. 11A and 12A, the chemiluminescent signal after fourminutes and 24 minutes, respectively, at various concentrations ofalkaline phosphatase for BZPD is shown. As clearly shown, BZPD offerssuperior detection sensitivities (i.e., a greater signal) even at verylow concentrations of enzyme (10⁻⁷ moles or less). As shown in FIGS. 11Band 12B, the S/N ratio of BZPD at low concentrations of alkalinephosphatase is comparable to previously developed dioxetanes whereas athigh alkaline phosphatase concentrations the S/N ratio of BZPD issuperior. This heightened sensitivity of BZPD can be used to detect verysmall amounts of material.

FIGS. 13 and 14 illustrate two heteroaryl substituted benzothiazoledioxetanes according to the invention. FIG. 13 shows the generalstructure of a 3-pyridyl substituted benzothiazole dioxetane accordingto a preferred embodiment of the invention. FIG. 14 shows the generalstructure of a 2-benzothiazolyl substituted benzothiazole dioxetaneaccording to a preferred embodiment of the invention. The substituent Xin FIGS. 13 and 14 can be a protecting group which can be removed bychemical or enzymatic means.

The emission spectrum for 3-pyridyl-BZPD was measured. The half life(t_(1/2)) of the 3-pyridyl-BZPD was found to be about 2 seconds. Themaximum emission wavelength (λ_(max)) of 3-pyridyl-BZPD was found to beabout 574 nm. This wavelength is red shifted approximately 24 nm fromthe maximum emission wavelength of BZPD which is approximately 550 nm.

This red shift observed for 3-pyridyl-BZPD can be used to advantage invarious applications. For example, in applications using a CCD camerafor detection, the red shift can enhance detection by the CCD. Further,in dual wavelength applications, the red shift of the 3-pyridyl-BZPD canbe used to increase sensitivity. For example, in dual wavelengthapplications where blue light emissions (e.g., λ_(max)=approximately 470nm) are used for one event and yellow-orange light (e.g.,λ_(max)=approximately 574 nm) from the decomposition of 3-pyridyl-BZPDis used for another event, greater separation between the two emissionspectra can be obtained. Greater separation between the emission spectracan result in greater detection sensitivity. Dual wavelength assays aredescribed in U.S. Pat. No. 4,931,223 which has been incorporated byreference above.

FIG. 15 shows the general structure of a second embodiment of aheteroaryl substituted benzothiazole dioxetane compound according to theinvention.

FIGS. 16 and 17 illustrate a synthesis route for the 3-pyridylsubstituted benzothiazole dioxetane shown in FIG. 13.

The 1,2-dioxetanes according to the invention can be used in a methodfor detecting the presence and amount of an analyte in a sample.Typically, the analyte will cause the dioxetane to decompose andgenerate light by cleaving the substituent X (as set forth in generalFormulae I and II above) from the dioxetane. According to the method ofthe invention, the sample along with the 1,2-dioxetane of the inventioncan be incubated and then inspected for the generation of light. Iflight is detected, the presence of the analyte which removes X isindicated. Further, the amount of light detected can be used todetermine the amount of the analyte present in the sample. Typically,the analyte is an enzyme, which is selected to cleave the substituent Xon the dioxetane. Upon removal of X, the dioxetane can decompose andgenerate light. According to the invention, the enzyme can be complexedto a biological moiety of interest.

The methods may be used in conjunction with the enhancement molecules,preferably the onium quaternary polymers and additives, discussed above,and set forth in U.S. Pat. No. 5,330,900 and 5,547,836. In preferredembodiments, the light emitted by the decomposition of the 1,2-dioxetaneof the invention is detected by a CCD camera.

The chemiluminescent substrates according to the invention can beprovided in a kit which includes the 1,2-dioxetanes of the inventioneither alone, or together with an enzyme or other substance which causesthe dioxetane to decompose by removing X. Chemiluminescent enhancers andadditives which improve the performance of the enhancers, can also beincluded in the kits.

The following example is a representative synthesis of benzothiazoledioxetane substrates and their precursors, and should not limit thescope of the claims. First, 4,6-dibromo-o-anisidine was obtainedaccording to the literature: Fuchs, Monatshefte fur Chemie, 36, 130,1915. A Varian Unity 300 NMR Spectrometer was used. All NMR data isproton (¹H) NMR.

EXAMPLE 1 2-Benzamido-3,5-dibromoanisole

The 4,5-dibromo-o-anisidine (11.3 g; 40.2 mmol), was dissolved in 75 mldichloromethane and 6.7 ml pyridine. The mixture was stirred at roomtemperature under argon. Benzoyl chloride (4.8 ml; 1.03 equivalents) wasadded dropwise by syringe. The mixture was stirred for 8 hours to obtainan orange-brown suspension. The reaction mixture was then concentratedto one-third volume on the rotary evaporator. The thick slurry wasfiltered on a Buchner funnel, washing the flask and solid with 50:50dichloromethane/hexanes. The resulting white solid was then washedliberally with water to remove any pyridine hydrochloride. The solidproduct was then dried in vacuo to obtain 13.56 grams of theabove-titled product. The biphasic filtrate was washed with water in afunnel, separating the organic layer, which was then rotary evaporatedto yield a purple brown solid. Trituration with 50:50dichloromethane/hexanes and recrystallization from minimal ethyl acetategave a second crop, weighing 1.43 grams. NMR(300 MHz/DMSO-d6) data wasas follows: δ 3.81(s, 3H), 7.36(1H), 7.44-7.71(m, 4H), 7.88-8.11(m, 2H)9.88(s, 1H). IR(CH₂Cl₂/cm⁻¹) data was as follows: 3420, 2980, 2940,1691, 1585, 1487, 1400, 1041, 875, 837.

EXAMPLE 2 N-(2,4-dibromo-6-methoxy)phenylthiobenzamide

The product of the preceding example (14.4 g; 37.4 mmol) was dissolvedin 35 ml dry pyridine with slight warming. Phosphorous pentasulfide (11g; 49.5 mmol) was added in portions under argon. A thick, light yellowcomplex formed exothermically. This mixture was stirred for 2 hours inan oil bath at 90° C. to give a thinner, darker yellow suspension. Themixture was then refluxed for 15 minutes and cooled to room temperature.The mixture was treated with 125 ml ethyl acetate to precipitate a gum.Water, 1 ml, was added with swirling to agglomerate the gum prior todecantation of the supernate. The gum was then triturated with 2×25 mlethyl acetate. The combined organics were rotary evaporated to yield anorange oil which contained pyridine. A 7% solution of sodium hydroxidein water was added to the oil with vigorous stirring for 20 minutes. Thesolution was filtered to remove insolubles, rinsing with minimalhydroxide solution. The filtrate was then acidified to pH 1 with 3M HClto precipitate a flocky, light yellow solid, which was dissolved in theminimal quantity of dichloromethane. The organic layer was separated androtary evaporated to yield 12.6 grams of the above-titled product as alemon-yellow solid. Analytical samples could be obtained byrecrystallization from ethanol to yield a one-spot material on TLC(Kieselgel 60-dichloromethane; Rf=0.56). NMR(300 MHz/DMSO-d6) data wasas follows: δ 3.81(s, 3H), 7.40-7.59(m, 6H), 7.90-7.93(m, 2H), 11.36(s,1H). IR(CHCl₃ /cm⁻¹) data was as follows: 3380, 2990, 1584, 1490, 1400,1345, 1040, 878, 838, 695.

EXAMPLE 3 2-Phenyl-4-methoxy-6-bromobenzothiazole

The thioamide from the preceding example (12.6 g; 31.4 mmol) was warmedin 30 ml of methanol. The suspension was swirled during the addition of7.35 ml of 4.3 M sodium methoxide in methanol (31.6 mmol). During theaddition, the solid dissolved and the yellow color faded to light amber.Rotary evaporation of the solvent and pumping in vacuo gave an amber,glassy solid which coated the glass. This thioamide salt was kept underargon during the addition of 20 ml of N-methylpyrrolidone. The flask wascapped with a septum and connected to a bubbler as it was placed in anoil bath at 110-120° C. Upon stirring for 30 minutes, a solid developedas the color became green-brown. The flask was then cooled toward roomtemperature before 100 ml of water was added to produce an off-whitesolid. The mixture was filtered, and the solid washed liberally withwater. After drying in vacuo, the solid was recrystallized from 50:50ethyl acetate:hexanes to yield 7.05 grams of white, hair-like needles.TLC showed the blue fluorescent product spot at Rf=0.47, while a traceUV absorbing impurity was present at a higher Rf (Kieselgel60-dichloromethane). The impurity could be removed by silica gelchromatography to obtain an analytical sample. NMR and IR data wereconsistent with the structure of the above-titled product. Inparticular, NMR(300 MHz/CDCl₃) data was as follows: δ 4.10(s, 3H),7.07(d, 1H), 7.497-7.52(m, 3H), 7.66(d, 1H), 8.11-8.14(m, 2H), andIR(CHCl₃/cm⁻¹) data was as follows: 3003, 2940, 1590, 1562, 1517, 1440,1400, 1387, 1322, 1260, 1055, 978, 830, 690.

EXAMPLE 4 2-Phenyl-4-methoxy-6-formylbenzothiazole

The chromatographed product from the preceding example (3g; 9.37 mmol)was dissolved in 70 ml of dry THE under argon. In another flask, 60 mldry THF was cooled and stirred at −78° under argon. To this flask, 5.6ml of 2.5 M n-Butyllithium (14.1 mmol) was added by syringe. Thesolution of bromobenzothiazole starting material was then added dropwiseunder argon from an addition funnel over 25 minutes. THF, 7 ml, was usedto rinse the funnel at the conclusion of the addition. The red-brownsolution was stirred for another 10 minutes at low temperature. Dry DMF,1.8 ml, was then added dropwise by syringe. After 10 minutes, thesolution was slowly warmed to room temperature over 1 hour. The reactionwas quenched by the rapid addition of 20 ml of 1 M aqueous ammoniumchloride solution. The THF was removed on the rotovap, and the productwas partitioned between ethyl acetate and the remaining water. The ethylacetate layer was washed four times with water to remove any DMF. Theorganics were dried over sodium sulfate and the solvent removed to yielda semi-solid paste. This was triturated with 20 ml of 20%dichloromethane in hexanes to yield a dry solid after decantation andpumping in vacuo. The resulting peach-colored product weighed 1.81 g TLCand showed essentially one spot at an Rf value of 0.62 (Kieselgel 60-10%ethyl acetate/hexanes). Spectral data for a similarly obtained productwere identical and consistent with that expected for the above-titledcompound. NMR(300 MHz/CDCI₃) data was: δ 4.18(s, 3H), 7.48-7.56(m, 4H),8.04(d, 1H), 8.17-8.20(m, 2H), 10.08(s, 1H). IR(CHCl₃/cm⁻¹) data was:3010, 2840, 2740,1695, 1595, 1572, 1480, 1470, 1395, 1290, 1270, 1145,1057, 983, 850, 690.

EXAMPLE 5 2-Phenyl-4-methoxy-6-formylbenzothiazole dimethyl acetal

Under argon, 1.8 grams of the aldehyde from the previous example (6.7mmol) was treated with 11 ml dichloromethane, 0.9 ml oftrimethylorthoformate, and 0.7 ml of anhydrous methanol. The suspensionwas stirred as 105 mg of toluenesulfonic acid monohydrate was added allat once. The flask was closed with a septum after purging it with argon.The solid soon dissolved to give a yellow-orange solution. Stirring wascontinued overnight at room temperature. The reaction mixture wasneutralized with excess triethylamine (0.15 ml) using a syringe. Themixture was stripped of all volatiles, dissolved in minimaldichloromethane, and plug-chromatographed on a 2 cm×1.5 inch column ofAlumina. The eluant was rotary evaporated and pumped to an oil whichslowly solidified. A sample was taken for immediate IR analysis, whichshowed the absence of any carbonyl absorption. This indicated thatacetal formation was complete, and the crude product was usedimmediately for the next reaction. IR(CH₂Cl₂/cm⁻¹) data was as follows:2940, 2840, 1602, 1580, 1468, 1410, 1355, 1198, 1155, 1060, 996, 837.

EXAMPLE 6Diethyl-1-methoxy-1-(2-phenyl-4-methoxybenzothiazol-6-yl)methanephosphonate

The crude product obtained in the previous step was dissolved in 11 mlof sieve-dried dichloromethane and 1.5 ml of triethylphosphite underargon. The flask was sealed with a septum, and the stirred solution wascooled to −78° C. in a dry ice/acetone bath. The pressure wasequilibrated at this temperature with an argon balloon. The mixture,which became a suspension, was then treated dropwise with 1.0 ml ofborontrifluoride etherate. The suspended solid dissolved as the contentswere slowly warmed to about −20° C. The solution was stored in therefrigerator for one hour and then slowly warmed to room temperature foran overnight stirring period. In the morning, 0.7 grams of solid sodiumbicarbonate was added, followed by 15 ml of saturated, aqueous sodiumbicarbonate solution. The biphase was stirred vigorously to expel carbondioxide. Water was added as necessary over 3 hours to dissolve anyinorganics. The dichloromethane layer was separated, and the aqueouslayer was back-extracted with 15 ml of the same solvent. The combinedorganics were subjected to TLC to show a single, UV/blue fluorescentspot at approximately Rf=0.15, tailing back to the origin (Kieselgel60-ethyl acetate). The solution was evaporated and vacuum pumped at 40°C. The viscous yellow oil was then dissolved in a minimal amount of50/50 dichloromethane/ethyl acetate and passed over a very short plug ofsilica gel. The eluant was stripped and chased several times with amixture of dichloromethane/hexanes. The oily product weighed 2.7 grams.NMR and IR spectroscopy showed a substantially pure product, but thepresence of moisture was indicated. NMR(300 MHz/CDCl₃) data was: δ1.21-1.36(m, 6H), 3.46(s, 3H), 3.93-4.21(m, 7H), 4.59-4.64(d, 1H),7.07(s, 1H), 7.47-7.52(m, 3H), 7.57(s, 1 H) 8.11-8.14(m, 2H).IR(CH₂Cl₂/cm⁻¹) data was: 3660 & 3460(H₂O), 2980, 2935, 2860, 1597,1570, 1510, 1480, 1460, 1445, 1408, 1342, 1245-1285(br), 1100, 1040(br),965(br), 865, 840, 610.

EXAMPLE 76-(Methoxytricyclo[3.3.1.1^(3,7)]dec-2-ylidenemethyl)-2-phenyl-4-methoxybenzothiazole

The pumped phosphonate ester from the previous step (2.7 g; 6.4 mmol)was dissolved in 25 ml of dry THF under argon. The solution was cooledto −78° C. with stirring in a flask outfitted with a septum and an argonballoon. The solution was treated dropwise with enough 2.5 M n-BuLi inhexanes to achieve a just permanent, red-purple color. In this process,all moisture and protic impurities have been titrated. Subsequently 2.7ml of the same n-BuLi solution (6.75 mmol) were added dropwise to yielda deep burgundy solution. After 10 minutes stirring at low temperature,2-adamantanone (0.95 grams, 6.33 mmol) was added as a solid under strongargon flow to exclude moisture. The solid dissolved over 10 minutes. Thesolution was then allowed to warm slowly to room temperature. A refluxcondenser was attached while maintaining an argon atmosphere. Themixture was refluxed for 1.5 hours to obtain a light orange solution.THF was then stripped on the rotary evaporator taking care to avoidfoaming. The product was partitioned between 25 ml ethyl acetate and 50ml 1:1 saturated sodium bicarbonate/water. The organic layer was thenwashed with 25 ml of water. The organic layer was dried over sodiumsulfate and stripped to yield a light yellow gum. The gum wasplug-chromatographed on a short column of silica gel, eluting withdichloromethane to remove trace polar contaminants. The appropriatefractions were pumped and chased with dichloromethane-hexanes. Thepumped product, weighing 2.14 grams, became a semi-solid upon storage inthe freezer. IR spectroscopy revealed a small 2-adamantanone carbonylband, indicating minor contamination which would be eliminated in thenext step. IR(CH₂Cl₂/cm⁻¹) data was as follows: 2920, 2850, 1597, 1567,1450, 1402, 1330, 1320, 1310, 1252, 1165, 1100, 1057, 978, 865, 640,620. Trace AD=0 at 1720 and 1710.

EXAMPLE 86-(Methoxytricyclo[3.3.1.1^(3,7)]dec-2-ylidenemethyl)-2-phenyl-4-hydroxybenzothiazole

A sodium ethanethiolate solution in DMF was made from 60% sodium hydrideand ethanethiol: 240 mg of 60% sodium hydride (6 mmol) was washed threetimes with hexanes under an argon atmosphere, removing the mineral oil.DMF, 11 ml, was added. The resulting suspension was cooled to 0° C. withstirring for the dropwise addition of ethanethiol (0.45 ml, 6 mmol).After hydrogen evolution ceased, the solution was warmed to roomtemperature and delivered by pipette to 1.64 grams ofmethoxy[2-phenyl-4-methoxy(benzothiazol-6-yl)methylidene adamantane in aseparate flask (3.9 mmol) under argon. The resulting solution wasstirred in an oil bath at 130° C. After one hour, the solution was deeporange and contained suspended solid. The reaction mixture was cooledand partitioned between 50 ml each of 1 M arnmonium chloride and 75%ethyl acetate/hexanes. The organic layer was washed 3 times with 25 mlof water. The combined aqueous layers were back-extracted with the samesolvent mixture, which was then washed several times with water. Thecombined organics were dried over sodium sulfate. TLC (Kieselgel60-dichloromethane) showed product at Rf=0.23, but also startingmaterial at Rf=0.39. Column chromatography (silica gel: 50%dichloromethane-hexanes to pure dichloromethane) allowed one purefraction of the lower Rf product to be isolated. Repeat chromatographyof the mixed fractions allowed additional product to be isolated. Afterstripping the solvents, a total of 245 mg of the above-entitled productwas obtained. NMR(300 MHz/CD₂Cl₂) data was as follows: δ 1.74-2.07(m,14H), 2.71(s, 1H), 3.26 (s, 1H), 3.32(s, 3H) 6.76(s, 1H), 6.94(d, 1H),7.38(d, 1H), 7.44-759(m, 3H), 7.99-8.16(m, 2H). IR(CH₂Cl₂/cm⁻¹)data was:3520, 2910, 2850, 1612, 1575, 1480, 1445, 1302, 1284, 1175, 1080, 980,860.

EXAMPLE 9 Disodium6-(methoxytricyclo[3.3.1.1^(3,7)]dec-2-ylidenemethyl)-2-phenylbenzothiazolyl-4-phosphate

Molecular sieve-dried pyridine, 4.0 ml, was placed in a flask underargon. The flask was outfitted with a magnetic stir bar and placed in anice bath. Distilled phosphorus oxychloride, 0.112 ml (1.2 mmol), wasadded dropwise by syringe. In another flask, 245 mg of thehydroxybenzothiazole derivative from the previous example was dissolvedin 15 ml of anhydrous THF under argon. The THF solution was then addedslowly and dropwise to the stirred solution of phosphorylating agent.During the addition, a precipitate developed. At the end of theaddition, the flask and syringe were rinsed with 2 ml of THF which wasalso added slowly to the reaction flask. The reaction mixture was thenwarmed to room temperature and stirred for three hours. A cotton-tippedneedle on a 20 ml syringe was used to draw up the supernate, leaving thepyridine hydrochloride behind. This supernate was added dropwise to asolution of 15 ml 0.5 M sodium hydroxide, which had been diluted to avolume of 75 ml with water, while being stirred at ice-bath temperature.The slightly cloudy solution cleared upon warming to room temperature.The solution was carefully pumped to remove THF and the volume adjustedto 110 ml with 5.0 ml acetonitrile and water. This solution was injectedin two portions onto a Polymer Laboratories 2 inch polystyrenereverse-phase HPLC column. A gradient of 5% to 10% acetonitrile was usedto allow separation of the major peak absorbing at 270 nm. This gradientwill require optimization for any specific equipment. The appropriatefractions were pooled and lyophilized to obtain 294 mg of a light yellowsolid. Spectral data were in concert with the above-titled structure. Ananalytical HPLC chromatogram on a similar support, using an acetonitrilegradient against 0.1% aqueous sodium bicarbonate, showed a singleproduct eluting at 13.2 minutes (approximately 50% acetonitrile). TheNMR(300 MHz/D₂0) data was: δ 1.38-2.02(m, 14H), 2.51(s, 1H), 3.00(s,1H), 3.24(s, 3H), 7.26-7.53(m, 5H), 7.75-8.04(m, 2H).

EXAMPLE 10 Disodium6-(4-methoxyspiro-[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3,7)]decan]-4-yl)2-phenylbenzothiazolyl-4-phosphate

In a tube was placed 285 milligrams of the enol ether phosphate from theprevious step. The solid was wet down with 1.0 ml of methanol and thendissolved by adding 25 ml of dichioromethane. The solution was thentreated with 0.5 ml of a solution of 5, 10, 15, 20-tetraphenyl-21 H, 23H-porphine (2 mg/ml in CHCl₃). The contents of the tube were cooled to0° C., while the solution was sparged with oxygen gas. After 5 minutes,while continuing to bubble oxygen through the solution, the tube wasirradiated with light from a cooled, 400 watt sodium vapor lamp whilemaintaining the temperature at 5° C. A 5 mil thick piece of Kaptonpolyimide film, placed between the lamp and the tube, filtered outunwanted UV radiation. The irradiation was continued for 17 minutes.Analytical HPLC [0.1% NaHC0₃ (H₂0)-acetonitrile gradient] showed thatthe conversion of the starting material, eluting at 13.2 minutes, to the1,2-dioxetane, which eluted at 12.94 minutes, was substantiallycomplete. The reaction mixture was stripped of solvents to give a redgum. Acetonitrile, 5 ml, and 0.05 M aqueous sodium hydroxide, 20 ml,were added with swirling and occasional cooling to dissolve the gum. DIwater was then added to give a total volume of 70 ml. This solution wasfiltered through highly retentive filter paper, rinsing carefully withwater in small portions, to give a filtrate volume of 110 ml. Thissolution was injected in two portions onto a Polymer Laboratories 1 inchpolystyrene reverse-phase HPLC column. A gradient of 5% to 100%acetonitrile (against water) was used to allow separation of the majorpeak absorbing at 270 nm. This gradient will require optimization forany specific equipment. An analytical HPLC chromatogram of the combinedproduct fractions on a similar support, using an acetonitrile gradientagainst 0.1% aqueous sodium bicarbonate, showed a single product elutingat 12.96 minutes (approximately 50% acetonitrile). The appropriatefractions were pooled and lyophillized to obtain 287 mg of a lightyellow solid. The product 1,2-dioxetane produced green light at 558 nmwhen triggered with alkaline phosphatase in an aqueous buffer at pH 8.5.UV: 213, 260.5, and 304 nm in 50/50 CH₃CN/H₂0.

EXAMPLE 11 2-Methoxy-4,6-dibromophenylisothiocyanate

In a flask under argon were placed 19.75 grams of4,6-dibromo-o-anisidine (70 mmol) and 20 grams of solid bicarbonate. Alarge, heavy-duty magnetic stir bar was added, followed by 120 mlacetonitrile and 50 ml dichloromethane. The suspension was stirred at 0°C. as 6.0 ml of thiophosgene was added rapidly by syringe. A thickprecipitate developed immediately. This was stirred vigorously as thecontents of the flask were slowly warmed to room temperature. The carbondioxide generated was led to a bubbler with a needle vent. The mixturethinned slightly as it warmed and was more easily stirred. Vigorousstirring was continued for two hours. The suspension was then recooledto 0° C. The solid was filtered off on a Buchner funnel, rinsing theflask and transferring any remaining solid with 30 ml of coldacetonitrile. The filtrate was rotary evaporated to a solid containingareas of orange discoloration. This solid was triturated with hexanes,pumped dry, and transferred to the Buchner funnel containing the white,original filter cake. This solid was washed with 5×100 ml portions of a0.5 M aqueous solution of NaH₂PO₄ in order to neutralize the inorganicbicarbonate present (carbon dioxide was released). The solid was brokenup during each rinse. The white product was then washed liberally withwater and dried in vacuo. The dry product weighed 21.8 grams. Analyticaldata obtained from a similarly synthesized product were in agreementwith the above-titled structure. NMR(300 MHz/CDCl₃) data was: δ 3.92(s,3H), 6.98(d, 1H), 7.31(d, 1H). IR(CH₂Cl₂/cm⁻¹) data was: 3020, 2970,2940, 2030(br), 1575, 1555, 1470, 1400, 1040, 935, 870, 840.

EXAMPLE 12 2-Phenyl-4-methoxy-6-bromobenzothiazole[One Pot Method]

About 15.5 grams of the isothiocyanate from the preceding example (48mmol) was dissolved in 50 ml of dry THF under argon. The solution wascooled to 0° C. with stirring in an ice bath. A solution ofphenylmagnesium bromide (Aldrich, 1.0 M in THF), 50 ml (50 mmol), wasadded by syringe in a thin stream. After stirring in the cold for 10minutes, the solution was slowly warmed to room temperature. At thispoint, a precipitate began to appear as a minor exotherm occurred. Thelight orange-brown suspension became thicker over 2 hours. The solventwas removed by rotary evaporation at 30° C. to obtain a moist,peach-colored solid coating the glass. This material was protected fromair as 50 ml of sieve-dried DMF was added. The solid dissolved with aslight exotherm. The flask was placed in an oil bath at 125° C. Over 45minutes, any residual THF was allowed to distill from the flask using ashort path distillation head. The mixture darkened during this time, anda suspended solid was produced. Upon cooling to room temperature, thecontents of the flask solidified. Aqueous 1 M HCl, 100 ml was added,breaking up the solid. Water, 100 ml, was added subsequently. Themixture was macerated to remove any coordinated magnesium ion. Themixture was then filtered and washed well with water. The moist solidwas taken up in 2×250 ml warm ethyl acetate, separating the supernatefrom insoluble flock. The combined organics were dried over sodiumsulfate and stripped to give 13.27 grams of a light brown solid. TLCshowed a major blue fluorescent product spot at Rf=0.47, while a traceUV absorbing impurity was present at a higher Rf (Kieselgel60-dichloromethane). A small, colored origin spot was removed by plugchromatography over silica gel (dichloromethane). Combining theappropriate fractions gave 12.29 grams of the product, essentiallyidentical to that of Example 3, but still containing a trace amount ofthe higher Rf impurity. The reaction of this example may also beacidified and worked up after the phenyl magnesium bromide has reactedwith the isothiocyanate to obtain the thioamide product of Example 2.

The following were also synthesized according to the general syntheticmethodology described above. One of skill in the art may easily invokeminor modifications as necessary. Any other route to the benzothiazolesystem may be employed as well.

EXAMPLE 13 2-(p-benzyloxybenzamido)-3,5-dibromoanisole

NMR(300 MHz/DMS0-d6):δ 3.79(s, 3H), 5.21(2H), 7.11-7.14(d, 2H),7.347-7.52(m, 7H), 7.93-7.96(d, 2H), 9.70(s, 1H).

EXAMPLE 14 N-(2,4-dibromo-6-methoxy)-p-benzyloxyphenylthiobenzamide

NMR(300 MHz/DMS0-d6):δ 3.80(s, 3H), 5.22(2H), 7.08-7.11(d, 2H),7.32-7.55(m, 7H), 7.95-7.98(d, 2H), 11.12(s, 1H).

EXAMPLE 15 2-(p-benzyloxy)phenyl-4-methoxy-6-bromobenzothiazole

NMR(300 MHz/DMS0-d6):δ 4.00(s, 3H), 5.21(2H), 7.17-7.22(m, 3H),7.34-7.50(m, 5H), 7.93-8.01(m, 3H).

EXAMPLE 16 2-(p-benzyloxy)phenyl-4-methoxy-6-formylbenzothiazole

NMR(300 MHz/DMSO-d6):δ 4.05(s, 3H), 5.23(2H), 7.20-7.23(d, 2H),7.33-7.50(m, 6H), 8.05-8.08(d, 2H), 8.31-8.32(d, 1H), 10.04(s, 1H).

EXAMPLE 17 2-(p-benzyloxy)phenyl-4-methoxy-6-formylbenzothiazoleDimethyl Acetal

IR(CHCl₃/cm⁻¹): 3000, 2940, 2840, 1611, 1580, 1530, 1490, 1470, 1355,1180, 1155, 1060, 1020, 980, 838, 700.

EXAMPLE 18diethyl-1-methoxy-1-[2-(p-benzyloxy)phenyl-4-methoxybenzothiazol-6-yl]methanephosphonate

NMR(300 MHz/CDCl₃):δ 1.19-1.37(m, 6H), 3.43(s, 3H), 3.86-4.18(m, 7H),4.51-4.75(m, 1H), 5.12(s, 2H), 7.02-7.03(m, 2H), 7.30-7.52(m, 7H),8.02-8.06(m, 2 H).

Example 196-(Methoxytricyclo[3.3.1.1^(3,7)]dec-2-ylidenemethyl-2-(p-benzyloxy)phenyl-4-methoxybenzothiazole

NMR(300 MHz/CDCl₃):δ 1.78-2.1(m, 14H), 2.74(s, 1H), 3.30(s, 1H), 3.35(s,3 H), 4.06(s, 3H), 5.12(s, 2H), 6.85-6.96(m, 1H), 6.99-7.12(m, 2H),7.29-7.50(m, 6H), 7.99-8.14(m, 2H).

TABLE 1 Half-life of Dephosphorylated dioxetanes pH 10. t_(1/2), secDioxetane BZPD 2.3 CSPD ® 57.6 CDP-Star ® 96 Plus Sapphire-II ™ BZPD54.9 CSPD ® 228 CDP-Star ® 420

1. A chemiluminescent compound of the general formula (II):

wherein Y is a heteroaryl group, X is a protecting group which can beremoved by chemical or enzymatic means, and R²-R⁶ are independentlyhydrogen, alkyl, substituted alkyl, aryl, or substituted aryl andwherein R³ and R⁴ may be joined as a spiro-fused cycloalkyl group. 2.The compound of claim 1, wherein X is a phosphate group.
 3. The compoundof claim 1, wherein the heteroaryl group Y is selected from the groupconsisting of pyridyl, benzothiozolyl, benzoxazolyl, benzofuranyl,benzothienyl, thiazolyl, isoxazolyl, isothiazolyl, quinolinyl, andpyrimidinyl.
 4. The compound of claim 3, wherein the heteroaryl group Yis a pyridyl selected from the group consisting of 2-pyridyl, 3-pyridyl,and 4-pyridyl.
 5. The compound of claim 3, wherein the heteroaryl groupY is 2-benzothiazolyl.
 6. The compound of claim 3, wherein theheteroaryl group Y is a thiazolyl selected from the group consisting of2-thiazolyl, 4-thiazolyl, and 5-thiazolyl.
 7. The compound of claim 3,wherein the heteroaryl group Y is an isoxazolyl selected from the groupconsisting of 3-isoxazolyl, 4-isoxazolyl, and 5-isoxazolyl.
 8. Thecompound of claim 3, wherein the heteroaryl group Y is an isothiazolylselected from the group consisting of 3-isothiazolyl, 4-isothiazolyl,and 5-isothiazolyl.
 9. The compound of claim 1, wherein the heteroarylgroup is substituted with one or more electron active groups.
 10. A kitfor detecting the presence of an analyte in a sample comprising: the1,2-dioxetane compound of claim 1; and an enzyme or enzyme conjugatewhich, in the presence of the dioxetane, causes the dioxetane todecompose and generate chemiluminescent emissions.
 11. The kit of claim10, wherein the substance comprises an enzyme.
 12. The kit of claim 10,further comprising a chemiluminescent enhancing agent which enhances thechemiluminescent emissions generated by the decomposition of thedioxetane.
 13. The kit of claim 12, wherein the chemiluminescentenhancing agent is a quaternary onium polymer.
 14. The kit of claim 12,further comprising an enhancement additive which further enhances thechemiluminescent emissions generated by the decomposition of thedioxetane.
 15. A kit for detecting the presence of an analyte in asample comprising: the 1,2-dioxetane compound of claim 2; and an enzymeor enzyme conjugate which, in the presence of said dioxetane, causessaid dioxetane to decompose and generate chemiluminescent emissions. 16.A method for detecting the presence and/or amount of an analyte in asample, comprising: adding the dioxetane compound of claim 1 to thesample, wherein the analyte is an enzyme or is conjugated to an enzymeand wherein the enzyme causes the dioxetane compound to decompose andgenerate light; incubating the sample; and inspecting the sample for thepresence of light; wherein the presence of light indicates the presenceof the analyte in the sample and wherein the amount of light indicatesthe amount of the analyte in the sample.
 17. The method of claim 16,further comprising adding a chemiluminescent enhancement agent to thesample to enhance the light generated.
 18. The method of claim 17,wherein the enhancement agent is a quatemary onium polymer.
 19. Themethod of claim 17, further comprising adding an enhancement additive tothe sample to further enhance the light generated.
 20. The method ofclaim 16, wherein the step of inspecting the sample comprises detectinglight with a CCD camera.
 21. The method of claim 16, wherein the analytecomprises an enzyme.
 22. The method of claim 21, wherein the enzyme iscomplexed with a biological moiety.
 23. A method for detecting thepresence and/or amount of two or more analytes in a sample, comprising:adding at least two different chemiluminescent compounds to the sample,at least one of which is the dioxetane compound of claim 1, wherein eachof the chemiluminescent compounds are configured to emit, upondecomposition, light of different wavelengths, and wherein each of theanalytes is an enzyme or is conjugated to an enzyme and wherein eachenzyme causes the corresponding chemiluminescent compound to decomposeand generate light of a particular wavelength; incubating the sample;and inspecting the sample for the presence of light; wherein thepresence of light emitted by one of the chemiluminescent compoundsindicates the presence of the corresponding analyte in the sample andwherein the amount of light emitted by one of the chemiluminescentcompounds indicates the amount of the corresponding analyte in thesample.
 24. The method of claim 23, wherein at least one of thechemiluminescent compounds added to the sample is the dioxetane compoundof claim 1 wherein the substituent Y is 3-pyridyl.
 25. The method ofclaim 24, wherein a second chemiluminescent compound added to the sampleemits light in the blue portion of the visible spectrum.
 26. The methodof claim 25, wherein the second chemiluminescent compound emits lighthaving a λ_(max) of approximately 470 nm.
 27. The compound of claim 3,wherein the heteroaryl group Y is 2-benzoxazolyl.
 28. The compound ofclaim 3, wherein the heteroaryl group Y is 2-benzofuranyl.
 29. Thecompound of claim 3, wherein the heteroaryl group Y is 2-benzothienyl.